Aryl phosphate derivatives of d4T having anti-HIV activity

ABSTRACT

Aryl phosphate derivatives of d4T with para-bromo substitution on the aryl group show markedly increased potency as anti-HIV agents without undesirable levels of cytotoxic activity. In particular, these derivatives are potent inhibitors of HIV reverse transcriptase. In a preferred aspect of the present invention, the phosphorus of the aryl phosphate group is further substituted with an amino acid residue that may be esterified or substituted, such as a methoxy alaninyl group.

This application is a Continuation of application Ser. No. 09/107,716,filed Jun. 29, 1998, now U.S. Pat. No. 6,030,957 which is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention is directed to aryl phosphate derivatives of2′,3′-didehydro-2′,3′-dideoxythymidine (hereinafter “d4T”) that exhibitpotent activity against the human immune deficiency virus (HIV), e.g. asinhibitors of HIV reverse transcriptase.

BACKGROUND OF THE INVENTION

The spread of AIDS and the ongoing efforts to control the responsiblevirus are well-documented. One way to control HIV is to inhibit itsreverse transcriptase activity (RT). Thus, novel, potent, and selectiveinhibitors of HIV RT are needed as useful therapeutic agents. Known,potent inhibitors of HIV RT include 5′-triphosphates of2′,3′-dideoynucleoside (“ddN”) analogues. These active RT inhibitors aregenerated intracellularly by the action of nucleoside kinase andnucleotide kinase. Thus ddN compounds such as AZT and d4T have beenconsidered to hold much promise in the search for anti-HIV agents.

The rate-limiting step for the conversion of 3′-azido-3′-deoxythymidine(Zidovudine; AZT) to its bioactive metabolite AZT-triphosphate seems tobe the conversion of the monophosphate derivative to the diphosphatederivative, whereas the rate-limiting step for the intracellulargeneration of the bioactive 2′,3′-dideoxy-2′,3′-didehydrothymidine (d4T)metabolite d4T-triphosphate was reported to be the conversion of thenucleoside to its monophosphate derivative.(Balzarini et.al., 1989, J.Biol. Chem. 264:6127; McGuigan et.al., 1996, J. Med. Chem. 39:1748). SeeFIG. 1 for the mechanism proposed in the prior art.

In an attempt to overcome the dependence of ddN analogues onintracellular nucleoside kinase activation, McGuigan et al. haveprepared aryl methoxyalaninyl phosphate derivatives of AZT (McGuiganet.al., 1993 J. Med. Chem. 36:1048; McGuigan et.al., 1992 Antiviral Res.17:311) and d4T (McGuigan et.al., 1996 J. Med. Chem.39:1748; McGuiganet.al., 1996 Bioorg. Med. Chem. Lett. 6:1183). Such compounds have beenshown to undergo intracellular hydrolysis to yield monophosphatederivatives that are further phosphorylated by thymidylate kinase togive the bioactive triphosphate derivatives in a thymidine kinase(TK)-independent fashion. However, all attempts to date to furtherimprove the potency of the aryl phosphate derivatives of d4T by varioussubstitutions of the aryl moiety without concomitantly enhancing theircytotoxicity have failed (McGuigan et.al., 1996 J. Med. Chem. 39:1748).

In the present invention, it has been discovered that a substitution atthe phenyl moiety in the phenyl methoxyalaninyl phosphate derivative ofd4T with an electron-withdrawing moiety such as a para-bromosubstitution, enhances the ability of the phenyl methoxyalaninylderivative of d4T to undergo hydrolysis due to the electron withdrawingproperty of the bromo substituent. The substituted phenyl phosphatederivative of d4T demonstrate potent and specific anti-viral activity.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected discovery that arylphosphate derivatives of d4T, for example having an electron-withdrawingsubstutution such as a para-bromo substitution on the aryl group, showmarkedly increased potency as anti-HIV agents without undesirable levelsof cytotoxic activity. In particular, these derivatives are potentinhibitors of HIV reverse transcriptase. In a preferred aspect of thepresent invention, the phosphorus of the aryl phosphate group is furthersubstituted with an amino acid residue that may be esterified orsubstituted such as a methoxy alaninyl group.

The para-bromo substituted phenyl methoxyalaninyl phosphate derivativeof d4T as an active anti-HIV agent potently inhibits HIV replication inperipheral blood mononuclear cells (PBMNC) as well as TK-deficient CEMT-cells without any detectable cytotoxicity. Furthermore, this novel d4Tderivative, d4T-5′-(para-bromophenyl methoxyalaninyl phosphate), hadpotent antiviral activity against RTMDR-1, an AZT- and NNI-resistantstrain of HIV-1, and moderate activity against HIV-2. Similarly, thecorresponding para-bromo substituted phenyl methoxyalaninyl phosphatederivative of AZT showed potent anti-HIV activity in PBMNC as well asTK-deficient CEM T-cells but it was not effective against the AZT- andNNI-resistant RTMDR-1 or HIV-2. In contrast to these d4T and AZTderivatives, the corresponding 3dT derivative, 3dT-5′-(para-bromophenylmethoxyalaninyl phosphate), showed no significant anti-HIV activity inPBMNC or TK-deficient CEM T-cells. To our knowledge, this is the firstreport of a previously unappreciated structure activity relationshipdetermining the potency of phenyl phosphate derivatives of both d4T andAZT.

The lead compounds d4T-5′-(para-bromophenyl methoxyalaninyl phosphate)and AZT-5′-(para-bromophenyl methoxyalaninyl phosphate provide a basisfor the design of effective HIV treatment strategies capable ofinhibiting HIV replication, and particularly in TK-deficient cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a prior art-proposed metabolic pathwayfor aryl phosphate derivatives of d4T.

FIGS. 2A and 2B are diagrams showing the electron withdrawing hypothesisfor the enhanced hydrolysis of a substituted phenyl ring.

FIG. 2C is an elution profile showing production of A-d4T as a result ofhydrolysis of each of the tested compounds: Compound 2, where X═H (opensquares); Compound 3, where X═OCH₃ (filled squares); and Compound 4,where X═Br (filled circles).

FIG. 2D is an elution profile showing the sensitivity of the testedcompounds to enzymatic hydrolysis by porcine liver esterase.

FIG. 3 is an elution profile showing the intracellular hydrolysis ofcompounds 2-4 in TK-deficient CEM cells. A metabolite peak withcorresponding to 680 pmols of A-d4T-MP was detected only in aliquotsfrom CEM cell lysates incubated with compound 4.

FIGS. 4A-4F show the chemical structures of compound 6c (FIG. 4A) andcompound 7c (FIG. 4B); the anti-HIV activity against HTLVIIIB in PBMNCand TK-deficient CEM T-cells for compound 6c(FIG. 4C) and for compound7c(FIG. 4D); and the antiviral activity against HIV-1 (HTLVIIIB), HIV-2and RTMDR-1 for compound 6c(FIG. 4E) and compound 7c (FIG. 4F).Antiviral activity was expressed as % inhibition of HIV replication asmeasured by RT activity in infected cells.

FIGS. 5A and 5B are schematic diagrams showing resonance effect(electron delocalization) of at the phenyl ring, whereby thepara-substituent and ortho-substituent of phenyl ring are expected tohave the same electronic effect.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered unexpectedly that certain derivatives of d4Tpossess increased activity against HIV while maintaining low levels ofcytotoxicity. As such, these derivatives are particularly useful asactive agents for anti-viral compositions, and for methods of treatingviral infections such as HIV infections.

Compounds of the Invention

The compounds of the invention, as discussed more fully in the Examplesbelow, are derivatives of d4T and AZT having potent antiviralactivities. Compounds substituted with an electron-withdrawing group,such as an ortho or para substituted halogen or NO₂ as shown in FIGS. 5Aand 5B, provide for more efficient hydrolysis to active inhibitorycompounds. Preferred is halogen substitution, and most preferred ispara-bromo substitution.

The d4T derivatives have aryl-phosphate substitution, with the arylgroup having an electron-withdrawing substitution, such as an ortho orpara-substitution with a halogen (Br, Cl, F, I) or with NO₂substitution. One example is shown below, where R₂ is an amino acidresidue that may be esterified or substituted, for example—NHCH(CH₃)COOCH₃ or pharmaceutically acceptable salts or esters thereof.

Synthesis of the d4T Derivatives

The d4T derivatives can be prepared as follows. d4T can be prepared fromthymidine by the procedures discussed in Mansuri, et al., 1989, J. Med.Chem. 32, 461, the disclosure of which is incorporated herein byreference. Appropriately substituted aryl phosphorochloridate can beprepared by the procedures discussed in McGuigan, et al., AntiviralRes., 1992, 17:311, the disclosure of which is incorporated herein byreference. The phosphorochloridate is added to a solution of d4T inanhydrous THF containing N-methylimidazole to form the desired product.

The d4T derivatives are administered to patients in the form of suitablecompositions containing the d4T or AZT derivative as an active agentalong with a pharmaceutically acceptable carrier, adjuvant or diluent.Sustained release dosage forms may be used if desired. The compositionsare administered to a patient in need of the anti-viral activity in asuitable anti-viral amount, for example, sufficient to inhibit the HIVreverse transcriptase and/or inhibit replication of HIV in a host cells.The dose is administered according to a suitable dosage regimen.

EXAMPLES

The invention will be explained further with reference to the followingexamples, which should not be considered to limit the invention.

Example 1 Synthesis and Characterization of d4T Derivatives

d4T 1 was prepared from thymidine following the procedure of Mansuriet.al., 1989 J. Med. Chem. 32, 461. Appropriately substituted phenylmethoxyalaninyl phosphorochloridates were also prepared according to themethod reported by McGuigan et al 1992 Antiviral Res., 17, 311.Compounds 2-4 were synthesized as outlined below in Scheme 1.

Scheme 1. Phenylmethoxyalaninyl phosphorochloridate was added to thesolution of d4T and 1-methylimidazole in anhydrous THF and the mixturewas stirred at room temperature for 5-6 hours. Work up of the reactionmixture furnished the required derivatives in good yields. Columnchromatography was applied to obtain pure compounds.

Physical data of the synthesized compounds was determined by HPLC wasconducted by using C18 4×250 mm LiChrospher column eluted with 70:30water/acetonitrile at the flow rate of 1 ml/minute. The purity of thefollowing compounds exceeded 96% by HPLC. ¹³C NMR peaks labeled by starsare split due to diastereomers.

Compound 2 : yield: 81% ; IR (Neat): 3222, 2985, 2954, 1743, 1693, 1593,1491, 1456, 1213, 1153, 1039, 931, 769 cm⁻¹; ¹H NMR (CDCl₃) δ9.30 (br s,1H), 7.30-7.10 (m, 6H), 6.85-6.82 (m, 1H), 6.36-6.26 (m, 1H), 5.91-5.85(m, 1H), 5.00 (br m, 1H), 4.19-3.68 (m, 4H), 3.72, 3.71 (s, 3H), 1.83,1.80 (d, 3H), 1.38-1.25 (m, 3H); ¹³C NMR(CDCl₃) δ173.9, 163.7, 150.7,149.7, 135.7*, 133.2*, 129.6*, 127.3*, 125.0*, 120.0, 111.1, 89.6*,84.5*, 66.9*, 52.5*, 50.0*, 20.9 and 12.3; ³¹P NMR(CDCl₃) δ2.66 , 3.20;MALDI-TOF mass m/e 487.9 (M+Na); HPLC retention time: 5.54 & 5.85minutes.

Compound 3: yield: 92%; IR (Neat): 3223, 3072, 2999, 2953, 2837, 1743,1693, 1506, 1443, 1207, 1153, 1111, 1034, 937, 837 and 756 cm⁻¹; ¹HNMR(CDCl₃) δ9.40 (brs, 1H), 7.30-7.00 (m, 5H), 6.83-6.81 (m, 1H),6.37-6.27 (m, 1H ), 5.91-5.86 (m, 1H), 5.00 (br m, 1H), 4.40-4.30 (m,2H), 4.20-4.10 (m, 2H), 3.95-3.93 (s, 3H), 3.82-3.80 (s, 3H), 1.85-1.81(s, 3H) and 1.39-1.29 (m, 3H); ¹³C NMR(CDCl₃) δ174.0, 163.9, 156.6 ,150.8, 143.5, 135.8*, 133.3*, 127.4*, 121.2*, 114.5, 111.2, 89.7*, 84.5,66.9*, 55.5, 52.5, 50.6*, 20.9, and 12.3; ¹³P NMR(CDCl₃) δ3.82, 3.20;MALDI-TOF mass m/e 518.2 (M+Na); HPLC retention time: 5.83 & 6.26minutes.

Compound 4: yield: 83%; IR (Neat): 3203, 3070, 2954, 2887, 2248, 1743,1693, 1485, 1221, 1153, 1038, 912, 835, 733 cm⁻¹; ¹H NMR(CDCl₃)δ9.60-9.58 (br s, 1H), 7.45-7.42 (m, 2H), 7.30-7.09 (m, 4H), 6.37-6.27(m, 1H), 5.93-5.88 (m, 1H), 5.04-5.01 (br m, 1H), 4.35-4.33 (m, 2H),4.27-3.98 (m, 2H), 3.71-3.70 (s, 3H), 1.85-1.81 (s, 3H), 1.37-1.31 (m,3H); ¹³C NMR(CDCl₃) δ173.7, 163.8, 150.8, 149.7*, 135.6*, 133.1*,127.4*, 121.9*, 118.0, 111.2*, 89.7*, 84.4*, 67.8*, 52.5, 50.0*, 20.7,and 12.3; ³¹P NMR(CDCl₃) δ3.41, 2.78; MALDI-TOF mass m/e 567.1 (M+Na);HPLC retention time: 12.04 & 12.72 minutes.

Example 3 Susceptibility of Compounds 2-4 to Hydrolysis

FIGS. 2A and 2B show a schematic representation of the electroniceffects of the para substituent in the phenyl ring of metaboliteprecursor B (see FIG. 1). To assess the susceptibility of compounds tohydrolysis, Compounds 2-4 were dissolved in methanol and then treatedwith 0.002 N NaOH. The concentrations were kept constant and thegeneration of the hydrolysis product A-d4T-MP was monitored using HPLC.A Lichrospher column (C18) was used for the HPLC runs. The column waseluted under isocratic conditions using the solvent mixture 70:30water/acetonitrile, and the elution profile is shown in FIG. 2C.

Hydrolysis of compounds was tested in a porcine liver esterase system.The data are shown in FIG. 2C. Compounds 2 and 4 (1 mM in Tris-HCl) wereincubated with 100 U of porcine liver esterase (Sigma) in Tris-HClbuffer (pH 7.4) for 2 hours at 37° C. Reaction, was stopped by addingacetone and chilling the reaction mixture. Following centrifugation at15,000×g, 0.1 mL aliquots of the reaction mixture were examined for thepresence of the active metabolite A-d4T-MP by using a quantitativeanalytical HPLC method capable of detecting 50 pmols of the metabolite.The 0.1 mL aliquot of the reaction product of compound 4 contained 1.4nmols of A-d4T-MP, wheras no metabolite was detected in the reactionproduct of compound 2.

As shown in FIGS. 2A and 2B, the presence of an electron withdrawingsubstituent at the para position of the phenyl moiety is likely toincrease the hydrolysis rates of the phenoxy group in the metaboliteprecursor B (FIG. 2A and 2B) generated by the carboxyesterase-dependentfirst step (FIG. 1, A to B) of the metabolic pathway of phenyl phosphatederivatives of d4T. A single bromo substitution at the para position ofthe phenyl ring would not interfere with the recognition and hydrolysisof this compound by the carboxyesterase (Step A to B in FIG. 1). Anelectronic effect induced by the electron-withdrawing para-bromosubstituent would result in enhanced hydrolysis of phenoxy group Cyielding D and subsequently E, the precursors of the key metaboliteA-d4T-MP. In order to test this hypothesis, we compared theunsubstituted compound 2, para-methoxy (OCH₃) substituted compound 3,and para-bromo substituted compound 4(=d4T-5′-[p-bromo-phenylmethoxyalaninyl phosphate] or d4T-pBPMAP), fortheir rate of chemical hydrolysis after treatment with 0.002 N NaOH bymeasuring the generation of alaninyl-d4T-monophosphate (A-d4T-MP).

As shown in FIG. 2C, compound 4 with a para-bromo substitent showed amuch faster hydrolysis rate than the unsubstituted compound 2, whereascompound 3 with the electron donating substituent —OCH₃ at para positionhad a slower hydrolysis rate than either of those two compounds.Similarly, the lead compound 4 was more sensitive to enzymatichydrolysis by porcine liver esterase than compound 2 (FIG. 2D).

Example 4 Intracellular Metabolism of Compounds 2-4 in TK-deficient CEMCells

To analyze the intracellular metabolism of compounds 2-4 in TK-deficientcells, 1×10⁶ CEM cells were incubated with compounds 2-4 (100 μM) for 3hours and subsequently examined the formation of the partiallyhydrolyzed phosphate diester metabolite, alaninyl d4T monophosphate byHPLC. Notably, the amount of this metabolite in CEM cells treated withcompound 4 was substantially greater than in CEM cells treated withcompound 2 or 3 ((680 pmol/10⁶ cells vs<50 pmol/10⁶ cells; FIG. 3).

CEM cells were cultured in a medium composed of RPMI, 10% fetal bovineserum, and 1% penicillin/streptomycin. Ten million cells at a density of10⁶ cells/mL were incubated with 100 μM of these compounds for 3 hoursat 37° C. After incubation, cells were washed twice with ice-cold PBS,and extracted by addition of 0.5 mL of 60% methanol. Cell lysates werekept at −20° C. overnight, after which lysates were centrifuged at15000×g for 10 minutes to remove the cell debris. One hundred μLaliquots of these lysates were injected directly to HPLC. The HPLCsystem consisted of a Hewlett Packard (HP) 1100 series equipped with aquartemary pump, an auto sampler, an electronic degasser, a diodearraydetector, and a computer will a chemstation software program for dataanalysis. The samples were eluted on a 250×4.6 mm Sulpelco LC-DB C18column. A solvent gradient was utilized to resolve the metabolite fromthe parent compound, which consisted of a mixture of methanol and 10 mMammonium phosphate (pH 3.7). The gradient ran at a flow rate of 1mL/minute from 5 to 35% methanol for the first 10 minutes, kept at 35%methanol for 5 minutes, and famished with a linear gradient from 35 to100% methanol in the next 20 minutes. The detection wavelength was setat 270 nm. A metabolite peak with a retention time of 8.7 minutescorresponding to 680 pmols of A-d4T-MP was detected only in aliquotsfrom CEM cell lysates incubated with compound 4.

Because of its enhanced susceptibility to hydrolysis, compound 4 waspostulated to be a more potent anti-HIV agent than the other compounds.Compounds 2-4 as well as the parent compound d4T (1) were tested fortheir ability to inhibit HIV replication in peripheral blood mononuclearcells and TK-deficient CEM T-cells using previously described procedures(Zarling et.al., 1990 Nature 347:92; Erice et.al., 1993 Antimicrob.Agents Chemother. 37:835; Uckun et. al., 1998 Antimicrob. AgentsChemother. 42:383). Percent inhibition of viral replication wascalculated by comparing the p24 and RT activity values from the testsubstance-treated infected cells with those from untreated infectedcells. In parallel, the cytotoxicity of the compounds was examined usinga microculture tetrazolium assay (MTA) of cell proliferation, asdescribed in the Zarling, Enrice, and Uckun articles Supra).

The similarity of the IC₅₀ values for inhibition of HIV-1 replicationshown in Table 1, provide evidence that the d4T-aryl phosphatederivatives were not more potent than the parent compound d4T whentested in HIV-1-infected peripheral blood mononuclear cells. In accordwith previous reports, the ability of d4T to inhibit HIV-1 replicationwas substantially reduced in TK-deficient CEM cells. Whereas the IC₅₀value for inhibition of p24 production by d4T was 18 nM in peripheralblood mononuclear cells, it was 556 nM in TK-deficient CEM cells.Similarly, the IC₅₀ value for inhibition of RT activity increased from40 nM to 2355 nM (Table 1). While all 3 aryl phosphate derivatives weremore potent than d4T in TK-deficient CEM cells, compound 4(d4T-5′-[p-bromo phenylmethoxyalaninyl phosphate]) having a para-bromosubstituent in the aryl moiety, was 12.6-fold more potent in inhibitingp24 production (IC₅₀ values: 44 nM vs 556 nM) and 41.3-fold more potentin inhibiting the RT activity (IC₅₀ values: 57 nM vs 2355 nM) than d4T(Table 1).

TABLE 1 PBMNC CEM IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ Compound X [p24] [RT][MTA] [p24] [RT] [MTA] 1 (= d4T) 0.018 0.040 >10 0.556 2.355 >10 2 H NDND >10 0.145 0.133 >10 3 —OCH₃ 0.033 0.033 >10 0.106 0.320 >10 4 Br0.022 0.042 >10 0.044 0.057 >10

None of the tested compounds exhibited any detectable cytotoxicity toperipheral blood mononuclear cells or CEM cells at concentrations ashigh as 10,000 nM, as determined by MTA. Intriguingly, compound 3 with apara-methoxy substituent in the aryl moiety was 5.6-fold less effectivethan compound 4 in inhibiting the RT activity in HIV-infectedTK-deficient CEM cells (IC₅₀ values: 320 nM vs 57 nM) although these twocompounds showed similar activity in peripheral blood mononuclear cells(IC₅₀ values: 33 nM vs 42 nM). Thus, the identity of Thepara-substituent appears to affect the anti-HIV activity of the arylphosphate derivatives of d4T in TK-deficient cells. To our knowledge,this is the first demonstration that the potency as well as theselectivity index of the d4T-aryl-phosphate derivatives can besubstantially enhanced by introducing a single para-bromo substituent inthe aryl moiety. This previously unknown structure-activity relationshipdetermined by the aryl moiety of the phosphate derivatives of d[4Tprovides a basis for the design of potentially more potent d4Tanalogues.

Example 5 Activity of Compound 4 and AZT in MDR Cells

The activity of compound 4 (d4T-5′-[p-bromophenyl methoxyalaninylphosphate]) against HIV-MDR cells was compared withAZT-5′-[p-bromophenyl methoxyalaninyl phosphate] (P-AZT) and with AZT.The incubation and analysis methods used were as described above forExample 4.

As shown in Table 2, P-AZT and AZT have similar activities with the IC₅₀values of 1.5 and 2.0 nM, respectively. The activity of Compound 4 (0.02nM) is 100-fold more effective than AZT (2.0 nM).

TABLE 2

HIV-2 HIV-MDR IC₅₀ IC₅₀ Compound [RT] [RT] 4 0.4 0.02 P-AZT 3.9 1.5 AZT2.4 2.0

Example 6 Synthesis of Arylphosphate Derivatives of 3dT

By way of further comparison, the effect on anti-HIV activity of varioussubstitutions in the aryl group of arylphosphate derivatives of3′-deoxytbymidine (3dT) was studied. As shown in Scheme 2, 3dt 5 wasprepared from d4T 1 which was prepared from thymidine using theliterature procedure (Mansuri et al., 1989 J. Med. Chem. 32:461-466).Hydrogenation of 1 was carried out in ethanol in the presence of H₂ andcatalytic amount of 5% Pd/C to afford 3dT 5 in 85% yield. Appropriatelysubstituted phenyl methoxyalaninyl phosphorochloridates were alsoprepared according to the method reported by McGuigan et.al., 1992Antiviral Res 17:311-321, and compounds 6-11 were synthesized asoutlined in Scheme 2.

Scheme 2. The appropriately substituted phenyl methoxyalaninylphosphorochloridate was added to a mixture of 3dT and 1-methylimidazolein anhydrous THF. The reaction mixture were stirred for 12 h at roomtemperature and then solvent was removed. The resulting gum wasre-dissolved in chloroform and washed with 1 M HCl, saturated sodiumbicarbonate solution (except in the case of the NO₂ derivative) and thenwith water. The organic phase was dried by MgSO₄ and the solvent wasremoved in vacuo. The crude product was purified by silica gel flashcolumn chromatography eluted with 5% methanol in chloroform to give purecompounds 6-11 in good yields.

Physical data of the synthesized compounds was determined. HPLC wasconducted using C18 4×250 mm LiChrospher column eluted with 70:30water/acetonitrile at the flow rate of 1 ml/minute. The purity of thefollowing compounds exceed 96% by HPLC. ¹³C NMR peaks labeled by starsare split due to diastereomers.

Compound 5: yield: 85%; ¹H NMR(CDCl₃) δ11.1 (br s, 1H), 7.82 (s, 1H),5.97-5.94 (m, 1H), 5.10 (br s, 1H), 4.05-3.95 (m, 1H), 3.72-3.52 (m,2H), 2.30-1.86 (m, 4H), 1.77 (s, 3H); ¹³C NMR(CDCl₃) δ163.9, 150.4,136.4, 108.7, 84.8, 81.4, 62.2, 31.8, 25.1, and 12.5.

Compound 6: yield: 96%; IR (neat): 3211, 2955, 2821, 1689, 1491, 1265,1211, 1153, 1043 and 933 cm⁻¹; ¹H NMR(CDCl₃) δ10.1 (br s, 1H), 7.47 (s,1H), 7.32-7.12 (m, 5H), 6.14-6.08 (m, 1H), 4.41-4.21 (m, 4H), 4.05-4.00(m, 1H), 3.70, 3.69 (s, 3H), 2.37-2.32 (m, 1H), 2.05-1.89 (m, 7H),1.38-1.35 (dd, 3H); ¹³C NMR(CDCl₃) δ173.6*, 163.8, 150.3, 150.1*, 135.2129.4*, 124.7, 119.8*, 110.5*, 85.7*, 78.3*, 67.2*, 52.3, 50.1*, 31.6*,25.4*, 20.7*, and 12.4*; ³¹P NMR(CDCl₃) δ2.82 & 3.11; MS (MALDI-TOF):490.4 (M+Na); HPLC retention time=6.86, 7.35 minutes.

Compound 7: yield: 96%; IR (neat): 3217, 2954, 2821, 1743, 1689, 1489,1265, 1217, 1153, 1092, 1012, 926 & 837 cm⁻¹; ¹H NMR(CDCl₃) δ9.40 (br s,1H), 7.43-7.41 (m, 1H), 7.30-7.14 (m, 4H), 6.13-6.07 (m, 1H), 4.39-4.00(m, 5H), 3.71,3.70 (s, 3H), 2.38-2.36 (m, 2H), 2.09-1.89 (m, 5H),1.39-1.36 (dd, 3H); ¹³C NMR(CDCl₃) δ173.6*, 163.7, 150.2, 148.8*, 135.3,129.5-129.0, 121.5-121.3, 116.3, 110.6, 86.0*, 78.4*, 67.7*, 52.6*,50.2*, 31.8*, 25.4*, 20.9* and 12.5; ¹³P NMR(CDCl₃) δ2.87 & 3.09; MS(MALDI-TOF): 524.9 (M+Na); HPLC retention time=14.05, 14.89 minutes.

Compound 8: Viscous oil, yield: 96%; λ_(max): 223 (ε3338) and 269(ε4695) nm; IR (neat): 3211, 2955, 1743, 1693, 1500, 1569, 1265, 1197,1153, 1045, 923 & 843 cm⁻¹; ¹H NMR(CDCl₃) δ9.40 (br s, 1H), 7.45-7.43(d, 1H), 7.19-7.01 (m, 4H), 6.14-6.06 (m, 1H), 4.39-3.97 (m, 5H), 3.71,3.70 (s, 3H), 2.38-1.89 (m, 7H), 1.39-1.35 (t, 3H); ¹³C NMR(CDCl₃)δ173.6*, 163.7, 150.2, 150.1*, 135.3, 121.5*, 116.3*, 110.6*, 85.9*,78.4*, 67.7*, 52.6, 50.2*, 31.8*, 25.6*, 20.9*, and 12.5; ³¹P NMR(CDCl₃)δ3.13 & 3.37; MS (MALDI-TOF): 508.2 (M+Na); HPLC retention time=8.38,8.80 minutes.

Compound 9: yield: 83%; IR (neat): 3211, 2954, 1743, 1689, 1485, 1265,1217, 1153, 1010, 923 & 833 cm⁻¹; ¹H NMR(CDCl₃) δ9.82 (br s, 1H),7.45-7.41 (m, 3H), 7.15-7.11 (m, 2H), 6.14-6.06 (m, 1H), 4.39-4.00 (m,5H), 3.71, 3.70 (s, 3H), 2.38-1.89 (m, 7H), 1.39-1.35 (dd, 3H); ¹³CNMR(CDCl₃) δ173.6*, 163.8, 150.3, 148.5*, 135.2, 132.6*, 121.8*, 117.7,110.6*, 85.9*, 78.3*, 67.2*, 52.5, 50.2*, 31.6*, 25.6*, 20.8*, and 12.5;³¹P NMR(CDCl₃) δ2.83 & 3.05; MS (MALDI-TOF): 570.0 (M+2+Na); HPLCretention time=15.50, 16.57 minutes.

Compound 10: yield, 87%; IR (neat): 3203, 2955, 1743, 1684, 1593, 1522,1348, 1265, 1153, 1101, 920 &860 cm⁻¹; ¹H NMR(CDCl₃) δ9.51 (br s, 1H),8.24-8.21 (m, 2H), 7.42-7.37 (m, 3H), 6.13-6.08 (m, 1H), 4.39-4.03 (m,5H), 3.72, 3.71 (s, 3H), 2.38-1.89 (m, 7H), 1.41-1.38 (dd, 3H); ¹³CNMR(CDCl₃) δ173.4*, 163.7, 155.2*, 150.2, 144.4, 135.3, 125.9-125.4,120.6*, 115.4, 110.6*, 86.1*, 78.4*, 68.1*, 52.7, 50.2*, 31.7*, 25.8*,20.9* and 12.5; ³¹P NMR(CDCl₃) δ2.60 & 2.81; MS (MLDI-TOF): 535.0(M+Na); HPLC retention time=8.12, 10.14 minutes.

Compound 11: yield, 100%; IR (neat): 3209, 2954, 1743, 1506, 1468, 1265,1207, 1153, 1036, 937 & 835 cm⁻¹; ¹H NMR(CDCl₃) δ9.89 (br s, 1H),7.49-7.47 (m, 1H), 7.16-7.11 (m, 2H), 6.84-6.80 (m, 2H), 6.15-4.09 (m,1H), 4.39-4.02 (m, 5H), 3.77, 3.76 (s, 3H), 3.74, 3.73 (s, 3H),2.38-1.89 (m, 7H), 1.38-1.33 (t, 3H); ¹³C NMR(CDCl₃) δ173.7*, 163.9,156.3, 150.3, 143.7*, 135.2, 120.7*, 114.3*, 110.5, 85.7*, 78.4*, 67.3*,55.4, 52.4, 50.1*, 31.8*, 25.4*, 20.8* and 12.4* ; ³¹P NMR(CDCl₃) δ3.27& 3.52; MS (MALDI-TOF): 521.3 (M+1+Na); HPLC retention time=7.15, 7.66minutes.

Example 7 Antiviral Activity of 3dT Compounds 6-11

Compounds 6-11 as well as the parent compound 3dT were tested inside-by-side comparison with d4T for their ability to inhibit HIV-1replication in peripheral blood mononuclear cells and TK-deficient CEMT-cells using previously described procedures (Zarling et al., 1990;Erice et al., 1993; Uckun et al., 1998, Supra).

3dT as well as its derivatives were less active than d4T in peripheralblood mononuclear cells as well as TK-deficient CEM T-cells (Table 3).Notably, in peripheral blood mononucleare cells, the IC₅₀[RT] values forcompounds 6-11 were higher than the IC₅₀[RT] value of 3dT (1.2-3.1versus 0.7, Table 3), suggesting that these prodrugs are sufficientlystable and TK-independent steps in their metabolism, perhaps theirenzymatic hydrolysis, may be rate-limiting for generation of activespecies. In contrast, aryl phospate derivatives of d4T were reported tobe more potent than d4T suggesting that the TK-dependent generation ofd4T monophospate is rate-limiting in its metabolic activation (McGuiganet al., 1996a). In accordance with the reported results in theliterature regarding the biologic activity of aryl phospate derivativesof d4T and AZT (McGuigan et al., 1993, 1996a), the aryl phosphatederivatives of 3dT were more active than the parent compound 3dT ininhibiting HIV-1 replication in TK-deficient cells, albeit with stillhigh micromolar IC₅₀[RT] values (Table 3).

Since compounds 6-11 were less active in TK-deficient CEM T-cells thanthey were in peripheral blood mononuclear cells (PBMNC), it waspostulated that the conversion of 3dT monophosphate generated from theseprodrugs into its active triphosphate occurs at a much slower rate inthe absence of TK. By comparison, the aryl phospate derivatives of d4Tshowed similar activity in normal and TK-deficient cells (McGuigan etal., 1996 Bioorg.Med.Chem.Lett. 6:1183-1186).

Anti-HIV Activity of aryl phosphate derivatives of 3′-deoxythymidine(6-11) in normal peripheral blood mononuclear cells (PBMNC) andTK-deficient CEM T-cells. All data are in μM and representconcentrations required to inhibit viral replication, as measured byassays of RT activity, by 50% (IC₅₀ [RT])⁹ or the 50% cytotoxicconcentration, as measured by MTA(IC₅₀[MTA]) (Mansuri et.al., 1989 J.Med. Chem. 32:461).

TABLE 3 PBMNC CEM IC₅₀ IC₅₀ IC₅₀ IC₅₀ Compound X [RT] [MTA] [RT] [MTA] 6 H 2.1 >100 7.5 >100  7 Cl 2.1 >100 21.9 >100  8 F 3.1 >100 32.7 >100 9 Br 1.2 >100 22.8 >100 10 NO₂ 2.0 >100 22.6 >100 11 OMe 1.3 >10019.7 >100 3dT — 0.7 >100 91.2 >100 d4T — 0.004 >100 2.335 >100

As shown in FIGS. 5A and 5B, the electronic effect of the parasubstitutions in the phenyl ring should affect the hydrolytic conversionof B to D in the metabolic pathway of aryl phospate derivatives of 3dTdepicted in FIG. 1. The presence of an electron-withdrawing substituentat the para position of the phenyl moiety was expected to increase thehydrolysis rates of the substituted phenoxy groups in compounds 7-10(FIGS. 2A and 2B). However, these compounds were not more active thancompound 6 with no para substitution or compound 11 with an electrondonating para substituent, prompting the hypothesis that thecarboxyesterase-dependent first hydrolysis step in their metabolism (Ato B in FIG. 1) plays a critical and rate-limiting role for thegeneration of active 3dT metabolites. Thus, compounds 7-10 may serve asrelatively poor substrates for the putative carboxyesterase responsiblefor their hydrolysis according to metabolic pathway proposed for arylmethoxyalaninyl phosphate derivatives of nucleoside analogs (McIntee etal., 1997 J. Med. Chem. 40:3323-3331).

In summary, the aryl phospate derivative of 3dT did not behave as whatmight have been expected from published work regarding the metabolismand activity of the prodrug forms of a very similar nucleoside analog,d4T. Surprisingly, the aryl phospate derivatives of 3dT did rot elicitpromising anti-HIV activity in HIV-1 infected normal peripheral bloodmononuclear cells or TK-deficient CEM T-cell line.

Example 8 Anti-HIV Activity of Derivatives of d4T, AZT, and 3dT

As shown in Scheme 1, d4T 1 was prepared from thymidine using theliterature procedure (Mansuri et.al., 1989, Supra). Hydrogenation of 1in ethanol in the presence of H₂ and catalytic amount of 5% Pd/Cafforded 3dT 3 in 85% yield (Scheme 1).

AZT 2 was prepared from thymidine using the literature methods (Chuet.al., U.S. Pat. No. 4,841,039). The ddN phosphorylation agentspossessing different substituents in their phenoxy moieties 5a, 5b and5c were prepared from the commercially available phenols in two-stepprocedures (Scheme 2) (McGuigan et.al., 1992, Supra), where Compounds4a, 4b, 5a, 5b, 7a and 7b were previously reported. Compounds 4c and 5care novel and their synthetic procedures as well as charaterization dataare reported below.

The synthesis of phenyl methoxyalaninyl phosphate derivatives of d4T 1,AZT 2 or 3dT 3 was carried out by following the literature condition asshown in Scheme 3.(McGuigan et.al., 1992). The general syntheticprocedures are as follows: The appropriately substituted phenylmethoxyalaninyl phosphorochloridate 5 was added to a mixture of thedesired ddN (1, 2 or 3) and 1-methylimidazole in anhydrous THF. Thereaction mixture were stirred for 12 hours at room temperature and thensolvent was removed. The resulting gum was re-dissolved in chloroformand washed with 1M HCl, saturated sodium bicarbonate solution and thenwith water. The organic phase was dried by MgSO₄ and the solvent wasremoved in vacuo. The crude product was purified by silica gel flashcolumn chromatography using a solvent mixture of methanol and chloroformfor elution to give the desired pure compounds in good yields.

p-Bromophenyl phosphorodichloridate 4c. Following the proceduredescribed by McGuigan et al.,1993, Supra, a solution of p-bromophenol(13.20 g; 76.30 mmol) and distilled triethylamine (10.65 mL) inanhydrous Et₂O (165 mL) was added dropwise into a vigorously stirredsolution of phosphoryl chloride (8.5 mL; 91.2 mmol) in anhydrous Et₂O(83 mL) at 0° C. over a period of three hours under nitrogen atmosphere.Subsequently, the resultant mixture was gradually warmed up to roomtemperature, stirred efficiently overnight at room temperature and thenheated to reflux for two hours. The reaction mixture was cooled to roomtemperature and filtered under aspirator pressure. The precipitate waswashed with anhydrous Et₂O (2×50 mL). The combined Et₂O layers wereevaporated to dryness on rotary evaporator to yield crude 4c as a paleyellow oil which was then subjected to vacuum distillation to give pure4c (14.05 g; 63.5% yield) as a colorless viscous oil (bp. 110-115° C./2mm Hg). IR (Neat) 3095, 1481, 1303, 1187, 948, 829 cm⁻¹. ¹H NMR (300MHz, CDCl₃) δ7.50 (2H, d, J=9.0 Hz), 7.15 (2H, d, J=9.0 Hz). GC/MS (m/e)290 (M⁺), 254 (M⁺—Cl), 173 (M⁺—POCl₂, ⁸¹ Br), 171 (M⁺—POCl₂,⁷⁹Br), 156(M⁺—PO₂Cl₂, ⁸¹Br), 154 (M⁺—PO₂Cl₂, ⁷⁹Br).

p-Bromophenyl methoxyalaninyl phosphorochloridate 5c. Following theprocedure described by McGuigan et al.,Supra, a solution of distilledtriethylamine (8.80 mL; 63.14 mmol) in anhydrous CH₂Cl₂ (180 mL) wasadded dropwise via an addition funnel into a vigorously stirred solutionof p-bromophenyl phosphorodichloridate 4c (8.69 g; 29.97 mmol) andL-alanine methyl ester hydrochloride (4.19 g; 30.02 mmol) in anhydrousCH₂Cl₂ (250 mL) at −70° C. over a period of three hours under nitogenatmosphere. Subsequently, the resultant mixture was allowed to graduallywarm up to room temperature and stirred overnight at room temperature.The solvent was removed on rotary evaporator. Anhydrous Et₂O (300 mL)was added to dissolve the residue and then filtered under aspiratorpressure to remove the white solid. The white solid was rinsed withanhydrous Et₂O (2×60 mL). The Et₂O layers were combined and evaporatedto dryness to afford a quantitative yield of 5c (10.7 g) as a palepink-yellow viscous oil. This product was then used for the next stepreaction without further purification. IR (Neat) 3212, 2989, 2952, 1747,1483, 1270, 1209, 1147, 927, 831, 757 cm⁻¹. ¹H NMR (300 MHz, CDCl₃)δ8.70 (1H, br, Ala-NH), 7.48 (2H, d, J=9.0 Hz, aryl H), 7.16 (2H, d,J=9.0 Hz, aryl H), 3.79 & 3.77 (3H, s & s, —OCH₃), 1.51 & 1.40 (3H, d &d, Ala-CH₃). MS (CI, m/e) 357.9 (M⁺ ⁸¹Br), 355.9 (M⁺, ⁷⁹Br), 322.0(M⁺—Cl, ⁸¹Br), 320.0 (M⁺—Cl, ⁷⁹Br), 297.9 (M⁺—COOCH₃, ⁸¹Br), 295.9(M⁺—COOCH₃, ⁷⁹Br), 184.0 (M⁺—BrC₆H₄O).

Characterization data of phenyl methoxyalaninyl phosphate derivatives ofAZT 1, d4T 2 and 3dT 3: HPLC was conducted by using C18 4×250 mmLiChrospher column eluted with 70:30 water/acetonitrile at the flow rateof 1 ml/minute. The purity of the following compounds exceed 96% byHPLC. ¹³C NMR peaks labeled by asterisks were split due to diastereomersarising from the phosphorus stereocenters.

Compound 6a: yield: 81%; IR(Neat): 3222, 2985, 2954, 1743, 1693, 1593,1491, 1456, 1213, 1153, 1039, 931, 769 cm⁻¹; ¹H NMR (CDCl₃) δ9.30 (br s,1H), 7.30-7.10 (m, 6H), 6.85-6.82 (m, 1H), 6.36-6.26 (m, 1H), 5.91-5.85(m, 1H), 5.00 (br m, 1H), 4.19-3.68 (m, 4H), 3.72, 3.71 (s, 3H), 1.83,1.80 (d, 3H), 1.38-1.25 (m, 3H); ¹³C NMR(CDCl₃) δ173.9, 163.7, 150.7,149.7, 135.7*, 133.2*, 129.6*, 127.3*, 125.0*, 120.0, 111.1, 89.6*,84.5*, 66.9*, 52.5*, 50.0*, 20.9 and 12.3; ³¹P NMR(CDCl₃) δ2.66, 3.20;MALDI-TOF mass m/e 487.9 (M+Na); HPLC retention time: 5.54 & 5.85minute.

Compound 6b: yield: 92%; IR (Neat): 3223, 3072, 2999, 2953, 2837, 1743,1693, 1506, 1443, 1207, 1153, 1111, 1034, 937, 837 and 756 cm⁻¹; ¹HNMR(CDCl₃) δ9.40 (br s, 1H), 7.30.-7.00 (m, 5H), 6.83-6.81 (m, 1H),6.37-6.27 (m, 1H ), 5.91-5.86 (m, 1H), 5.00 (br m, 1H), 4.40-4.30 (m,2H), 4.20-4.10 (m, 2H), 3.95-3.93 (s, 3H), 3.82-3.80 (s, 3H), 1.85-1.81(s, 3H), and 1.39-1.29 (m, 3H); ¹³C NMR(CDCl₃) δ174.0, 163.9, 156.6,150.8, 143.5, 135.8*, 133.3*, 127.4*, 121.2*, 114.5, 111.2, 89.7*, 84.5,66.9*, 55.5, 52.5, 50.6*, 20.9, and 12.3; ³¹P NMR(CDCl₃) δ3.82, 3.20;MALDI-TOF mass m/e 518.2 (M+Na); HPLC retention time: 5.83 & 6.26minute.

Compound 6c: yield: 83%; IR (Neat): 3203, 3070, 2954, 2887, 2248, 1743,1693, 1485, 1221, 1153, 1038, 912, 835, 733 cm⁻¹; ¹H NMR(CDCl₃)δ9.60-9.58 (br s, 1H), 7.45-7.42 (m, 2H), 7.30-7.09 (m. 4H), 6.37-6.27(m, 1H), 5.93-5.88 (m, 1H), 5.04-5.01 (br m, 1H), 4.35-4.33 (m, 2H),4.27-3.98 (m, 2H), 3.71-3.70 (s, 3H), 1.85-1.81 (s, 3H), 1.37-1.31 (m,3H); ¹³C NMR(CDCl₃) δ173.7, 163.8, 150.8, 149.7*, 135.6*, 133.1*,127.4*, 121.9*, 118.0, 111.2*, 89.7*, 84.4*, 67.8*, 52.5, 50.0*, 20.7,and 12.3; ³¹P NMR(CDCl₃) δ3.41, 2.78; MALDI-TOF mass m/e 567.1 (M+Na);HPLC retention time: 12.04 & 12.72 minute.

Compound 7c: yield: 95%; IR (Neat) 3205.7, 3066.3, 2954.5. 2109.8,1745.3, 1691.3, 1484.9, 1270.9, 1153.2, 1010.5 and 926.1 cm⁻¹. ¹H NMR(300 MHz, CDCl₃) δ8.69 (1H, br, 3-NH), 7.45 (2H, d, J=9.0 Hz, aryl H),7.34 & 7.32 (1H, s & s, vinyl H), 7.11 (2H, d, J=9.0 Hz, aryl H), 6.18 &6.13 (1H, t & t, J=6.6 & 6.6 Hz, H at C-1′), 4.44-3.77 (6H, m, H atC-3′, 4′ & 5′, Ala-NH and Ala-CH). 3.73 & 3.72 (3H, s & s, —COOCH₃),2.51-2.20 (2H, m, H at C-2′), 2.18 (3H, s, —CH₃ at C-5), 1.39 & 1.36(3H, d & d, Ala-CH₃). ¹³C NMR (75 MHz, CDCl₃) δ173.6, 163.6 150.1,149.2, 149.1, 135.2, 132.4, 121.6, 117.8, 111.1, 85.0, 84.7, 81.9, 81.8,65.5, 60.1, 59.9, 52.4, 50.0, 49.9, 36.9, 20.6, 20.5, 12.2. MS (CI, m/e)589.1 (M⁺, ⁸¹ Br) and 587.1 (M⁺, ⁷⁹Br).

Compound 8a: yield: 96%; IR (Neat): 3211, 2955, 2821, 1689, 1491, 1265,1211, 1153, 1043 and 933 cm⁻¹; ¹H NMR(CDCl₃) δ10.1 (br s, 1H), 7.47 (s,1H), 7.32-7.12 (m, 5H), 6.14-6.08 (m, 1H), 4.41-4.21 (m, 4H), 4.05-4.00(m, 1H), 3.70, 3.69 (s, 3H), 2.37-2.32 (m, 1H), 2.05-189 (m, 7H),1.38-1.35 (dd, 3H); ¹³C NMR(CDCl₃) δ173.6*, 163.8, 150.3, 150.1*, 135.2,129.4*, 124.7, 119.8*, 110.5*, 85.7*, 78.3*, 67.2*, 52.3, 50.1*, 31.6*,25.4*, 20.7*, and 12.4*; ³¹P NMR(CDCl₃) δ2.82 & 3.11; MS (MALDI-TOF):490.4 (M+Na); HPLC retention time=6.86, 7.35 minute.

Compound8b: yield, 100%; IR(Neat): 3209, 2954, 1743, 1506, 1468, 1265,1207, 1153, 1036, 937 & 835 cm⁻¹; ¹H NMR(CDCl₃) δ9.89 (br s, 1H),7.49-7.47 (m, 1H), 7.16-7.11 (m, 2H), 6.84-6.80 (m, 2H), 6.15-6.09 (m,1H), 4.39-4.02 (m, 5H), 3.77, 3.76 (s, 3H), 3.74, 3.73 (s, 3H),2.38-1.89 (m, 7H), 1.38-1.33 (t, 3H); ¹³C NMR(CDCl₃) δ173.7*, 163.9,156.3, 150.3, 143.7*, 135.2, 120.7*, 114.3*, 110.5, 85.7*, 78.4*, 67.3*,55.4, 52.4, 50.1*, 31.8*, 25.4*, 20.8* and 12.4*; ³¹P NMR(CDCl₃) δ3.27 &3.52; MS (MALDI-TOF): 521.3 (M+1+Na); HPLC retention time=7.15, 7.66minute.

Compound 8c: yield: 83%; IR (Neat): 3211, 2954, 1743, 1689, 1485, 1265,1217, 1153, 1010, 923 & 833 cm⁻; ¹H NMR(CDCl₃) δ9.82 (br s, 1H),7.45-7.41 (m, 3H), 7.15-7.11 (m, 2H), 6.14-6.06 (m, 1H), 4.39-4.00 (m,5H), 3.71, 3.70 (s, 3H), 2.38-1.89 (m, 7H), 1.39-1.35 (dd, 3H); ¹³CNMR(CDCl₃) δ173.6*, 163.8, 150.3, 148.5*, 135.2, 132.6*, 121.8*, 117.7,110.6*, 85.9*, 78.3*, 67.2*, 52.5, 50.2*, 31.6*, 25.6*, 20.8*, and 12.5;³¹P NMR(CDCl₃) δ2.83 & 3.05; MS (MALDI-TOF): 570.0 (M+2+Na); HPLCretention time=15.50, 16.57 minute.

Cellular Assays of Anti-HIV Activity and Cytotoxicity. Anti-HIVactivities were evaluated in AZT-sensitive HIV-1(strain: HTLVIIIB)-,AZT- and NNI-resistant HIV-1(strain: RTMDR-1)-(kindly provided by Dr.Brendan Larder, NIH AIDS Research and Reference Reagent Program, DIV.AIDS, NIAID, NIH; cat. # 2529), or HIV-2(Strain: CBL-20)-infectedperipheral blood mononuclear cells (PBMNC) as well as HTLVIIIB-infectedTK-deficient CEM T-cells by determining the concentration of compoundneeded to inhibit viral replication by 50%, based on reversetranscriptase activity assays (IC₅₀ [RT]). Percent viral inhibition wascalculated by comparing the RT activity values from the testsubstance-treated infected cells with RT values from untreated infectedcells (i.e., virus controls). The 50% cytotoxic concentrations of thecompounds (CC₅₀[MTA]) were measured by microculture tetrazolium assay(MTA), using 2,3-bis(2-methoxy4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide (XTI) (Zarling et.al., 1990; Erice et.al., 1993, Uckun et.al.,1998, Supra).

Identification of d4T-5′-para-bromophenyl methoxyalaninyl phosphate) andAZT-5′-(para-bromophenyl methoxyalaninyl phosphate) as Potent anti-HIVagents

The d4T-phenyl phosphate derivatives were not more potent than theparent compound d4T when tested in HIV-1-infected PBMNC. The ability ofd4T to inhibit HIV-1 replication was substantially reduced inTK-deficient CEM cells. Whereas the IC₅₀ value for inhibition of the RTactivity by d4T was 40 nM in PBMNC, it was 2400 nM in TK-deficient CEMcells (Table 4 & FIGS. 4A-4F). While all three phenyl phosphatederivatives were more potent than d4T in TK-deficient CEM cells,compound 6c (d4T-5′-[p-bromo phenylmethoxyalainyl phosphate]) with apara-bromo substituent in the phenyl moiety was 60-fold more potent ininhibiting the RT activity (IC₅₀ values: 60 nM vs 2400 nM) than d4T(Table 4).

None of the compounds exhibited any detectable cytotoxicity to PBMNC orCEM cells at concentrations as high as 10,000 nM, as determined by MTA.Intriguingly, compound 6b with a para-methoxy substituent in the phenylmoiety was 5-fold less effective than compound 6c in inhibiting the RTactivity in HIV-infected TK-deficient CEM cells (IC₅₀ values: 300 nM vs60 nM) although these two compounds showed similar activity inperipheral blood mononuclear cells (IC₅₀ values: 30 nM vs 40 nM) (Table4).

Compounds 7a, 7b, 7c and their parent compound AZT 2 were tested fortheir ability to inhibit HIV replication in PBMNC and TK-deficient CEMT-cells (Table 4). Percent inhibition of viral replication wascalculated by comparing the RT activity values from the testsubstance-treated infected cells with those from untreated infectedcells. In parallel, the cytotoxicity of the compounds was examined usinga microculture tetrazolium assay (MTA) of cell proliferation. Theability of AZT 2 to inhibit HIV-1 replication was substantially reducedin TK-deficient CEM cells. Whereas the IC₅₀ value for inhibition of RTactivity by AZT was 3 nM in PBMNC, it was 200 nM in TK-deficient CEMcells. Unlike the corresponding d4T derivatives, the unsubstituted andpara substituted phenyl phosphate derivatives of AZT were not morepotent than the parent compound AZT when tested in HIV-1 infectedTK-deficient CEM T-cells. However, the para-bromo substituted phenylphosphate derivative of AZT, AZT-5′-para-bromophenyl methoxyalaninylphosphate) 7c, was 5 times more effective than AZT in inhibiting HIVreplication of TK-deficient CEM cells (IC₅₀ [RT] values: 0.04 μM vs 0.2μM). None of the compounds exhibited any detectable cytotoxicity toPBMNC or CEM cells at concentrations as high as 10,000 nM, as determinedby MTA.

Compounds 8a-c and their parent compound 3dT 3 were tested inside-by-side comparison with d4T 1 for their ability to inhibit HIV-1replication in PBMNC and TK-deficient CEM T-cells. 3dT as well as itsderivatives were less active than d4T in peripheral blood mononuclearcells as well as TK-deficient CEM T-cells (Table 4). Notably, inperipheral blood mononuclear cells, the IC₅₀[RT] values for compounds8a-c were higher than the IC₅₀[T] value of 3dT (1.2-3.1 versus 0.7,Table 4), suggesting that these prodrugs are sufficiently stable andTK-independent steps in their metabolism, perhaps their enzymatichydrolysis, may be rate-limiting for generation of active species. Inaccordance with the reported results in the literature regarding thebiologic activity of phenyl phospate derivatives of d4T and AZT thephenyl phosphate derivatives of 3dT were more active than the parentcompound 3dT in inhibiting HIV-1 replication in TK-deficient cells,albeit with still high micromolar IC₅₀[RT] values (Table 4 & FIGS.4A-4F). Since compounds 8a-c were less active in TK-deficient CEMT-cells than they were in PBMNC, we postulate that the conversion of 3dTmonophosphate generated from these prodrugs into its active triphosphateoccurs at a much slower rate in the absence of TK.

TABLE 4 Anti-HIV Activity of phenyl methoxyalaninyl phosphatederivatives of d4T, AZT and 3dT in normal peripheral blood mononuclearcells (PBMNC) and TK-deficient CEM T-cells.

PBMNC CEM IC₅₀ IC₅₀ IC₅₀ IC₅₀ Compound X [RT] [MTA] [RT] [MTA] 6a H N.D.N.D. 0.1 >10 6b OCH₃ 0.03 >10 0.3 >10 6c Br 0.04 >10 0.06 >10 7a H N.D.N.D. 1.7 >10 7b OMe 0.1 >10 4.1 >10 7c Br 0.004 >10 0.04 >10 8a H2.1 >10 7.5 >10 8b OMe 1.3 >10 19.7 >10 8c Br 1.2 >10 22.8 >10 1(d4T) —0.04 >10 2.4 >10 2(AZT) — 0.003 >10 0.2 >10 3(3dT) — 0.7 >10 91.2 >10

Activity of the Lead Compounds d4T-51-para-bromophenyl methoxyalaninylphosphate) and AZT-51-para-bromophenyl methoxyalaninyl phosphate)against 2HIV-2 and RTMDR-1

The lead compounds 6c and 7c were tested in side-by-side comparison withAZT 2 for their ability to inhibit HIV replication in RTMDR-1, and AZT-and NNI-resistant strain of HIV-1, and HIV-2 in PBMNC (Table 5). Thenovel d4T derivative 6c, d4T-5′-(para-bromophenyl methoxyalaninylphosphate), had potent activity against RTMDR-1 and moderate activityagainst HIV-2. However, the corresponding para-bromo substituted phenylmethoxyalaninyl phosphate derivative of AZT 7c and the parent AZT 2 werenot effective against the AZT resistant RTMDR-1 or against HIV-2.

TABLE 5 Anti-HIV Activity of lead compounds 6c and 7c in HIV-2 andRTMDR-1 cells.

HIV-2 RTMDR-1 IC₅₀ IC₅₀ Compound [RT] [RT] 6c 0.4 0.02 7c 3.9 1.5 2(AZT)2.4 2.0

All data are in μM and represent concentrations required to inhibitviral replication, as measured by assays of RT activity, by 50%(IC₅₀[RT]).

Compounds 6a, 6b and 6c are all more potent than the parent d4T 1 inTK-deficient CEM cells, while these d4T-phenyl phosphate derivatives(6a, 6b and 6c) are not more potent than the parent d4T 1 in HIV-1infected PBMNC (Table 4). Comparing all the phenyl methoxyalaninylphosphate derivatized d4T, d4T-5′-[p-bromo phenylmethoxyalaninylphosphate] 6c is the most potent anti-HIV agent in TK-deficient CEMcells. This observation could be attributed to the para-bromosubstituent in the phenyl moiety of 6c which enhances the ability of itsphosphorus to undergo hydrolysis due to the electron withdrawingproperty of the bromo substituent (FIG. 2) and results in generation ofsubstantially higher quantities of the key metabolite d4T monophosphatein the TK-deficient CEM T-cells (McIntee et.al., 1997, J. Med. Chem.40:3233-3331).

The potency of phenyl, methoxyphenyl and bromophenyl phosphatederivatives of AZT in TK-deficient CEM cells also followed the sametrend as that of d4T derivatives, namely 7c (bromophenyl) >7a(phenyl) >7b (methoxyphenyl). However, among the three phenylmethoxyalaninyl phosphate derivatives of AZT (7a, 7b and 7c), only 7cshowed higher potency than AZT in TK-deficient CEM cells (IC₅₀ values:40 nM vs 200 nM). For phenyl methoxyalaninyl phosphate derivatives of3dT (Table 4), the presence of an electron-withdrawing substituent atthe para position of the phenyl moiety was expected to increase thehydrolysis rates of the substituted phenoxy group in compound 8c (e.g. Bto C in FIG. 2). However, 8c was not more active than compound 8a withno para substitution or compound 8b with an electron donating parasubstituent, prompting the hypothesis that the carboxyesterase-dependentfirst hydrolysis step in their metabolism (e.g. A to B in FIG. 2) playsa critical and rate-limiting role for the generation of active 3dTmetabolites. We postulate that compounds 8a, 8b and 8c may serve asrelatively poor substrates for the putative carboxyesterase responsiblefor their hydrolysis according to metabolic pathway proposed for phenylmethoxyalaninyl phosphate derivatives of nucleoside analogs (FIG. 2).The aryl phospate derivatives of 3dT did not behave as what might havebeen expected from the published work regarding the metabolism andactivity of the prodrug forms of a very similar nucleoside analog, d4T.To much of our surprise, the aryl phospate derivatives of 3dT did notelicit promising anti-HIV activity in HIV-1 infected normal peripheralblood mononuclear cells or TK-deficient CEM T-cell line.

In summary, d4T-5′-[p-bromo-phenylmethoxyalaninyl phosphate] 6c andAZT-5′-p-bromo-phenylmethoxyalaninyl phosphate] 7c were identified asactive anti-HIV agents which potently inhibit HIV replication inTK-deficient CEM T-cells without any detectable cytotoxicity.Furthermore, the novel d4T derivative 6c had potent antiviral activityagainst RTNMDR-1, an AZT- and NNI-resistant strain of HIV-1, andmoderate activity against HIV-2. In contrast to these d4T and AZTderivatives, the corresponding 3dT derivative, 3dT-5′-(para-bromophenylmethoxyalaninyl phosphate), showed no significant anti-HIV activity inPBMNC or TK-deficient CEM T-cells. To our knowledge, this is the firstcomprehensive report of a previously unappreciated structure activityrelationship determining the potency of phenyl phosphate derivatives ofd4T and AZT. Further development of the lead compounds 6c and 7c mayprovide the basis for the design of effective HIV treatment strategiescapable of inhibiting HIV replication in TK-deficient cells.

While a detailed description of the present invention has been providedabove, the invention is not limited thereto. The invention describedherein may be modified to include alternative embodiments, as will beapparent to those skilled in the art. All such alternatives should beconsidered within the spirit and scope of the invention, as claimedbelow.

We claim:
 1. A compound of the formula:

or a pharmaceutically acceptable salt thereof.
 2. A method forinhibiting virus replication in a cell infected with virus, the methodcomprising administering to the infected cell a virus replicationinhibiting amount of a compound of the formula:

or a pharmaceutically acceptable salt thereof.
 3. The method of claim 2,wherein the virus comprises an HIV.
 4. A method for inhibiting virusreverse transcriptase in a cell infected with virus, the methodcomprising administering to the infected cell a reverse transcriptaseinhibiting amount of a compound of the formula:

or a pharmaceutically acceptable salt thereof.
 5. The method of claim 4,wherein the virus comprises an HIV.
 6. A pharmaceutical compositioncomprising an amount effective for inhibiting viral reversetranscriptase in an infected cell of a compound of the formula:

or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier, adjuvant or diluent.
 7. The composition of claim 6,wherein the virus comprises an HIV.
 8. A pharmaceutical compositioncomprising an amount effective for inhibiting virus replication in aninfected cell of a compound of the formula:

or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier, diluent, or adjuvant.